Drug combination for the treatment of viral diseases

ABSTRACT

This invention pertains to a method for treating a human with human immunodeficiency virus infection which comprises administering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor, or pharmaceutically acceptable salts thereof.

BACKGROUND OF THE INVENTION

[0001] This is a continuation application of U.S. patent applicationSer. No. 08/929,249 filed Sep. 10, 1997 (Lyon & Lyon Docket No.266/298), which is a continuation-in-part application of U.S. patentapplication Ser. No. 08/585,287 (Lyon & Lyon Docket No. 266/297), filedon Jan. 11, 1996, which is a continuation-in-part application of U.S.patent application Ser. No. 08/403,320 filed on Mar. 14, 1995 (Lyon &Lyon Docket No. 266/303), all entitled “New Drug Combination for theTreatment of Viral Diseases” by Strair et al, and all herebyincorporated by reference herein in its entirety, including thedrawings.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for treating a humanwith human immunodeficiency virus infection. The method comprisesadministering to the human a therapeutically effective amount of athymidine analog, which analog acts as an inhibitor of viral reversetranscriptase necessary for viral replication of human immunodeficiencyvirus, and a thymidylate synthase inhibitor. In other embodiments, themethod further comprises administering to the human a therapeuticallyeffective amount of a folate antagonist or hydroxyurea, or both.

DESCRIPTION OF THE BACKGROUND

[0003] The disclosures referred to herein to illustrate the backgroundof the invention and to provide additional detail with respect to itspractice are incorporated herein by reference. For convenience, thedisclosures are referenced in the following text and respectivelygrouped in the appended bibliography.

Sanctuary Growth of HIV in the Presence of AZT

[0004] Acquired immunodeficiency syndrome (AIDS) is believed to becaused by the human immunodeficiency virus (HIV). Human immunodeficiencyvirus is a retrovirus which replicates in a human host cell. The humanimmunodeficiency virus appears to preferentially attack helper T-cells(T-lymphocytes or OKT4-bearing T-cells). When the helper T-cells areinvaded by the virus, the T-cells become a human immunodeficiency virusproducer. The helper T-ells are quickly destroyed causing the B-cellsand other T-cells, normally stimulated by helper T-cells, to no longerfunction normally or produce sufficient lymphokines and antibodies todestroy the invading virus or other invading microbes.

[0005] Although the human immunodeficiency virus does not necessarilycause death, the virus generally causes the immune system to be sodepressed that the human develops secondary infections such as herpes,cytomegalovirus, pneumocystis carinni, toxoplasmosis, tuberculosis,other mycobacteria, and other opportunistic infections. Kaposi'ssarcoma, lymphomas, and cervical cancer may also occur. Some humansinfected with the human immunodeficiency virus appear to live withlittle or no symptoms, but appear to have persistent infections, whileothers suffer mild immune system depression with symptoms such as weightloss, malaise, fever, and swollen lymph nodes. These syndromes have beencalled persistent generalized lymphadenopathy syndrome (PGL) and AIDSrelated complex (ARC) and generally develop into AIDS. Humans infectedwith the AIDS virus are believed to be persistently infective to others.

[0006] Human immunodeficiency virus is an extremely heterogeneous virus.The clinical significance of this heterogeneity is evidenced by theability of the virus to evade immunological pressure, survive drugselective pressure, and adapt to a variety of cell types and growthconditions. A comparison of isolates among infected patients hasrevealed significant diversity, and within a given patient, changes inthe predominant isolate over time have been noted and characterized. Infact, each patient infected with human immunodeficiency virus harbors a“quasispecies” of virus with a multitude of undetected viral variantspresent and capable of responding to a broad range of selectivepressures, such as those imposed by the immune system or antiviral drugtherapy. Therefore, diversity is a major obstacle to pharmacologic orimmunologic centrol of human immunodeficiency virus infection. Humanimmunodeficiency virus infection has multiple mechanisms to maximize itspotential for genetic heterogeneity. These mechanisms result in anextremely diverse population of virus capable of responding to a broadrange of selective pressures, including the immune system andantiretroviral therapy, with the outgrowth of genetically altered virus.

[0007] When a patient with human immunodeficiency virus infection isinitiated on antiretroviral therapy, there is generally a virologicresponse characterized by declining virernia and antigenemia(5,7,19,20,25). Unfortunately, the currently available antiretroviralagents which have undergone clinical evaluation have only limitedbenefit because most patients will ultimately have evidence of worseningdisease and increasing viral burden.

[0008] This progression often occurs in association with the emergenceof drug-resistant human immunodeficiency virus. For example, mostpatients who are treated with 3′-azido-3′-deoxythymidine (AZI) will haveinitial evidence of improvement of clinical and laboratory parameters ofhuman immunodeficiency virus infection (7,20). The duration of thisbenefit varies from patient to patient and is likely to be disease stagerelated (21).

[0009] Ultimately, however, most patients will have progressive diseaseand genotypic or phenotypic evidence of the appearance of AZT-resistanthuman immunodeficiency virus (9,12). Since clinical failure and theappearance of virus with high level resistance to AZT both occur withevidence of increasing levels of viremia and changes in viral tropism,it has been difficult to ascribe the clinical failure solely to thedevelopment of AZT resistance (2,11). Nevertheless, it seems likely thatAZT resistance ultimately contributes to the clinical failure seen inmost patients receiving prolonged AZT therapy.

[0010] While the development of viral-encoded drug resistance maycontribute to the limited efficacy of currently used antiretroviralagents, it cannot explain all of the in vitro and in vivo phenomenaassociated with viral replication in the presence of an antiretroviralagent. For example, many patients will have continued evidence of viralreplication after initiation of AZT therapy, but the isolated virus willremain sensitive to AZT when analyzed in tissue culture (7,20). Incontrast, high level human immunodeficiency virus resistance to many ofthe non-nucleoside reverse transcriptase inhibitors develops veryrapidly in culture and in patients (13,16,22,23). Some of thesedifferences may relate to the complexity and prevalence of viralvariants harboring pre-existing drug resistance mutations prior to theapplication of the selective pressure. However, some of the differencesmay be due to cellular heterogeneity in the uptake or metabolism of theantiretroviral agents, that is, each cell population may have some cellsthat are refractory to the antiviral effects of the drug. This wouldallow a subset of the cellular population to be successfully infected bygenetically drug-sensitive human immunodeficiency virus in the presenceof the antiviral drug. Depending upon the prevalence of drug-resistanthuman immunodeficiency virus in the initial population, the relativerates of replication of drug-resistant and drug-sensitive virus, and thepercentage of cells refractory to the antiviral effects of the drug,different patterns of viral breakthrough would emerge. Notably, thenon-nucleoside reverse transcriptase inhibitors do not undergo cellularmetabolism and cellular effects of uptake or metabolism may be lesslikely in this setting. This is consistent with the observation thatviral-encoded drug resistance to the non-nucleoside reversetranscriptase inhibitors develops very rapidly under selection in tissueculture and in patients. In fact, the rapid development of resistance inpatients suggests that the blood and plasma compartment of virus issubjected to drug selective pressure. The presence of humanimmunodeficiency virus, but lack of AZT-resistant human immunodeficiencyvirus, early after the initiation of AZT suggests that a component ofthis viral pool may be capable of averting selective drug pressure invivo. Continued viral replication in cells in which AZT is anineffective antiretroviral agent could conceivably result in thecontinued growth of virus that is sensitive to AZT. An increase in thenumber of these cells over time could also alter viral growth kineticsin the presence of AZT without the emergence of virus with high levelAZT resistance. Therefore, many mechanisms may contribute to theinability of an antiviral agent to completely suppress humanimmunodeficiency virus infection. Viral growth in the presence of thenon-nucleoside reverse transcriptase inhibitors appears due to the rapidselection of genetically resistant virus. In contrast, genetic viraldrug resistance does not appear to be the major mechanism contributingto early viral growth in the presence of AZT.

[0011] The use of recombinant human immunodeficiency virus encodingreporter genes has been reported to analyze viral breakthrough infectionin the presence of antiretroviral agents (26). In that study, todetermine the prevalence of viral variants spontaneously resistant tothe non-nucleoside reverse transcriptase inhibitor TIBO R82150, HeLa-T4cells were infected in the presence of drug with replication defectiveHIV-gpt (18,26) or HIV-LacZ (26). The recombinant virus used for theseinfections was produced by infection of cell lines containing anintegrated copy of the defective recombinant virus withreplication-competent human immunodeficiency virus. Thereplication-competent human immunodeficiency virus provided thenecessary gene products to rescue the defective virus. The prevalence ofviral variants containing mutations encoding resistance to TIBO R82150was reflected by the prevalence of recombinant viruses capable ofinfecting HeLa-T4 cells in the presence of TIBO R82150. The presence ofreporter genes in the recombinant viruses allowed for a quantitativeanalysis of a single cycle of infection on a single cell basis.

[0012] U.S. Pat. No. 4,724,232 (Rideout et al.) discloses a method fortreating a human having acquired immunodeficiency syndrome whichcomprises administering to the human 3′-azido-3′-deoxythymidine.

[0013] Cancer, Dec. 15, 1992, vol. 70, no. 12, pp. 2929-2934 (Posner etal.) discloses the use of 3′-azido-3′-deoxythymidine and 5-fluorouracilin the treatment of cancer.

Early HIV Breakthrough Infection in the Presence of Stavudine

[0014] The measurement of plasma HIV RNA copy number after theinitiation of antiviral therapy has provided several insights into thekinetics and dynamics of HIV infection. Initial studies quantitating HIVRNA after the initiation of a non-nucleoside reverse transciptaseinhibitor. (NNRTI), nevirapine, indicated a very rapid turnover ofplasma HIV (28). In those studies there was an initial decline in HIVRNA followed by a rapid increase in plasma viral RNA. The studies withnevirapine demonstrated that the rapid rebound in HIV RNA levels was aconsequence of the outgrowth of HIV with phenotypic and genotypicresistance to nevirapine (28). An in vitro model of HIV infection afterthe initiation of a different NNRTI (TIBO) has also indicated asimilarly high prevalence of variants capable of infection in thepresence of the drug (26).

[0015] Similar clinical and laboratory studies analyzing early HIVinfection in the presence of AZT have also been undertaken (29). Incontrast to the clinical studies with nevirapine, early HIV infection inthe presence of AZT does not appear to be predominated by the earlyoutgrowth of drug-resistant HIV. While the amount of virus circulatingin plasma shortly after the initiation of AZT rapidly declines, theremaining circulating virus after this decline does not containmutations known to encode resistance to AZT (29). Laboratory infectionsusing an in vitro model of infection in the presence of AZT havedemonstrated a similar pattern: early breakthrough infection independentof the presence of genetic resistance (31). These more complex dynamicsmay be a consequence of a variety of pharmacologic, cellular and viralfeatures. The mutations associated with AZT-resistance may be present inthe initial (unselected) viral population but mutant HIV with high levelAZT-resistance generally contain multiple mutations associated withAZT-resistance and the emergence of these variants often occurs overmonths-years. While the slow emergence of these high level resistantvariants can be explained by a low prevalence of AZT-resistant variants,the need for superimposed mutations, or selection against the emergenceof these variants, the early outgrowth of AZT-sensitive virus in thepresence of AZT must be explained by virologic, cellular orpharmacologic features that result in the ability of HIV-1 that isgenotypically and phenotypically sensitive to AZT to replicate in thepresence of AZT.

[0016] A quantitative in vitro model of HIV infection which utilizesrecombinant HIV has been used to characterize some of the mechanismsresponsible for HIV kinetics after the initiation of antiviral drugs(26,31). In that model a replication-defective HIV encoding a selectablemarker is used to assess a single cycle of infection in the absence ofeither repeated cycles of infection or selection of virus in thepresence of antiviral drugs. The use of a replication-defective virusallows an assessment of mechanisms of early HIV breakthrough infectionin the presence of antiviral drugs and has been used to quantitate HIVbreakthrough infection. Similarly, this system has been used todetermine that such infection in the presence of a NNRTI is likely dueto infection by genetically resistant virus while early infection in thepresence of AZT is due to the infection by virus without genetic drugresistance (26,31). These in vitro results mimic those described inclinical studies of HIV dynamics after the initiation of a NNRTI (28) orAZT (29).

[0017] Another feature of the replication-defective recombinant HIVsystem is that cells infected with HIV in the presence of the antiviraldrug can be readily isolated and characterized. Using this approach ithas been possible to determine that some of the cells infected in thepresence of AZT had metabolic features that rendered AZT an ineffectiveantiviral drug. Attempts to reverse these metabolic features hasresulted in the development of new drug combinations designed tomodulate the antiviral efficacy of AZT. One such combination hasimproved antiviral efficacy in both cells demonstrated to be refractoryto the antiviral effects of AZT and primary blood mononuclear cells(35).

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 is a schematic representation of the production ofrecombinant HIV-gpt by COS cell transfection or rescue from theH9/HIV-gpt cell line.

[0019]FIG. 2 is a schematic representation of the analysis of coloniesarising after COS cell derived HIV-gpt infection of HeLa-T4 cells in thepresence of 10 μM AZT.

[0020]FIG. 3 is a graph showing the infection of a clone of HeLa-T4cells “print resistant” to the antiviral effects of AZT (clone R116) anda control clone (S1) with replication-competent HIV-1IIIB in thepresence of 0.1 μM AZT.

[0021]FIGS. 4A and 4B are graphs illustrating thymidine metabolism-HPLCanalysis of clones obtained after infection of HeLa-T4 cells withHIV-gpt in the presence and absence of AZT.

[0022]FIG. 5 is a graph showing a comparison of thymidine kinase MRNAlevels (A) and enzyme activity (B) in cell lines sensitive andpersistently resistant to the antiretroviral effects of AZT.

[0023]FIG. 6 is a graph showing cellular toxicity of AZT.

[0024]FIG. 7A and FIG. 7B are graphs showing the suppression of viralbreakthrough in cells sensitive and refractory to the antiviral effectsof AZT.

[0025]FIG. 8 is a graph illustrating FUdR cytotoxicity in cellssensitive and refractory to the antiretroviral activity of AZT.

[0026]FIG. 9 is a graph showing AZT-FUdR cytotoxicity in JE6.1 cellssensitive and resistant to the antiviral effects of AZT.

[0027]FIG. 10 is a graph showing that the AZT-FUdR combination inhibitsHIV-1 infection of PBMC.

[0028]FIG. 11 is a graph showing the infection of JE6.1 cell clonespersistently resistant to the antiviral effects of d4T (D4T bulk, D4TR1,D4TR3) and a control clone of JE6.1 cells with HIV-IIIB in the presenceof various concentrations of d4T.

[0029]FIG. 12 is a graph showing that the D4T-FUdr combination inhibitsHIV-1 infection of PBMCs.

SUMMARY OF THE INVENTION

[0030] This invention pertains to a method for treating a human withhuman immunodeficiency virus infection (acquired immunodeficiencysyndrome) which comprises administering to the human a therapeuticallyeffective amount of a thymidine analog, which analog acts as aninhibitor of viral reverse transcriptase necessary for viral replicationof human immunodeficiency virus, and a thymidylate synthase inhibitor,or pharmaceutically acceptable salts thereof. In other embodiments, themethod further comprises administering to the human a therapeuticallyeffective amount of a folate antagonist or hydroxyurea, or both.

DETAILED DESCRIPTION OF THE INVENTION Sanctuary Growth of HIV in thePresence of AZT

[0031] Human immunodeficiency virus resistance to the non-nucleosidereverse transcriptase inhibitors emerges very rapidly under selection inculture and in patients. In contrast, AZT-resistant HIV generallyemerges in patients only after more prolonged therapy. Although HIV canbe cultured from many patients shortly after the initiation of AZTtreatment, characterization of the virus that is cultured generallyindicates that it is sensitive to AZT. To initiate an evaluation of themechanisms contributing to early HIV breakthrough in the presence of AZTand other nucleoside analogs, replication-defective HIV encodingreporter genes were utilized. These recombinant HIV allow a quantitativeanalysis of a single cycle of infection. Results with these defectiveHIV indicate that early infection in the presence of AZT often resultsfrom the infection of a cell which is refractory to the antiretroviraleffects of AZT.

[0032] Characterization of cell lines derived from such infected cellshas demonstrated decreased accumulation of AZT, increasedphosphorylation of thymidine to TTP, and increased levels of thymidinekinase activity. In addition, AZT inhibition of replication-competentHIV infection is also significantly impaired in this cell line.

Early HIV Breakthrough Infection in the Presence of Stavudine

[0033] By utilizing replication-defective HIV encoding reporter genes,applicants have demonstrated that early HIV breakthrough infection inthe presence of Stavudine results from infection of cells which arerefractory to the antiviral effects of the drug. In addition, applicantsdemonstrate that the combination of Stavudine and Floxuridine has potentantiviral activity in cells refractory to the antiviral activity ofStavudine alone. Data is presented indicating that the predominantmechanism of HIV breakthrough early after the initiation of stavudine(d4T) is related to the inefficacy of d4T as an antiviral agent in asubset of the host population. This inefficacy is demonstrated to beindependent of the presence of HIV with genetic drug resistance and hasalso been demonstrated in several cell types and with retroviruses otherthan HIV. These results may help explain several features of the in vivoand in vitro selection of d4T resistant virus and contribute to anunderstanding of the mechanisms responsible for HIV kinetics after theinitiation of antiviral drugs.

[0034] The present invention relates to a method for treating a humanwith human immunodeficiency virus infection. The method comprisesadministering to the human a therapeutically effective amount of athymidine analog, which analog acts as an inhibitor of viral reversetranscriptase necessary for viral replication of human immunodeficiencyvirus, and a thymidylate synthase inhibitor. Thymidine analogs, such as3′-azido-3′-deoxythymidine (AZT), are prodrugs in the treatment ofacquired immunodeficiency syndrome. 3′-Azido-3′-deoxythymidine isconverted by cellular enzymes to 3′-azido-3′-deoxythymidinemonophosphate (AZTMP).

[0035] The monophosphate is then converted by cellular enzymes to3′-azido-3′-deoxythymidine diphosphate (AZTDP) and3′-azido-3′-deoxythymidine triphosphate (AZTTP). In human cells infectedwith HIV, 3′-azido-3′-deoxythymidine triphosphate is an inhibitor of theviral reverse transcriptase necessary for viral replication. Some cells,however do not efficiently metabolize AZT to the triphosphate and mayoverproduce the natural thymidine triphosphate, which competes with theantiviral activity of AZTIP. Studies have demonstrated that these cellscontribute to the early failure of the antiviral activity of AZT. Bycoadministering a thymidylate synthase inhibitor with the thymidineanalog, applicants have found that that the thymidine analog is a moreeffective inhibitor of HIV replication. The thymidylate synthaseinhibitor may function by resulting in lower levels of thymidinetriphosphate to compete with the phosphorylated thymidine analog reversetranscriptase inhibition.

[0036] In another embodiment, the method further comprises administeringto the human a therapeutically effective amount of a folate antagonisttogether with the thymidine analog and the thymidylate synthaseinhibitor to modulate the effects of the thymidine analog. In yetanother embodiment, the method further comprises administering to thehuman a therapeutically effective amount of hydroxyurea together withthe thymidine analog and the thymidylate synthase inhibitor to modulatethe effects of the thymidylate synthase inhibitor. In still yet anotherembodiment, both the folate antagonist and hydroxyurea may beadministered with the thymidine analog and the thymidylate synthaseinhibitor.

Use of Floxuridine to Modulate the Antiviral Activity of AZT

[0037] Recent clinical studies have demonstrated that early HIVreplication after initiation of AZT is generally a consequence of thereplication of AZT-sensitive virus (29). A prior in vitro analysis ofthis early breakthrough replication in the presence of AZT hasdemonstrated the infection of cells in which AZT was an ineffectiveantiviral agent (31). A metabolic characterization of these cells hasled to the development of a novel combination therapy designed topotentiate the antiviral efficacy of AZT. The present inventiondescribes the antiviral effects of the combination of floxuridine andAZT. This combination suppresses early viral breakthrough, lowers theIC₅₀ of AZT, and has particular antiviral efficacy in the subset ofcells that are infected with AZT-sensitive virus in the presence of AZT.The antiviral efficacy of this combination in peripheral bloodmononuclear cells suggests potential clinical utility.

[0038] In an attempt to explain the ability of genetically-sensitive HIVto replicate in the presence of AZT, applicants have initially utilizedrecombinant replication-defective HIV to quantitate infection in thepresence of AZT (31). In those studies, replication-defective HIVencoding a selectable marker was used to infect target cells in thepresence of 10 μM AZT. The cells infected with the defective HIV wereisolated by expression of the selectable marker. A subset of theseinfected cells was demonstrated to be readily infected with another HIVin the presence of 10 μM AZT. These cells were persistently refractoryto the antiviral effects of AZT and were demonstrated to have excessivephosphorylation of thymidine to TTP, increased thymidine kinase activityand decreased accumulation of AZTTP.

[0039] These data suggested that a component of early infection withAZT-sensitive HIV in the presence of AZT was a consequence of theinfection of cells which were refractory to the antiviral effects ofAZT. Some of these cells had metabolic factors resulting in reducedAZTTP/TTP ratios in the cells. These data also suggest that it may bepossible to overcome this reduced antiviral efficacy of AZT bybiochemical modulation of TTP pool sizes. One way to potentiallymodulate these cells is with fluoropyrimidines such as5-fluorodeoxyuridine (FUdR). These compounds are known to reducecellular thymidine pools by the inhibition of thymidylate synthase.

[0040] In the present invention, applicants demonstrate the suppressionof early HIV infection in the presence of AZT with FUdR. FUdR will beshown to potentiate the antiviral effects of AZT in whole cellpopulations (including peripheral blood mononuclear cells [PBMC]) aswell as in subsets of cells isolated by infection with recombinant HIVin the presence of AZT. Infection of these latter cells will be shown tobe extremely sensitive to combined AZT-FUdR therapy.

[0041] The term “prodrug”, as used herein refers to compounds whichundergo biotransformation prior to exhibiting their pharmacologicaleffects. The chemical modification of drugs to overcome pharmaceuticalproblems has also been termed “drug latentiation.” Drug latentiation isthe chemical modification of a biologically active compound to form anew compound which upon in vivo enzymatic attack will liberate theparent compound. The chemical alterations of the parent compound aresuch that the change in physicochemical properties will affect theabsorption, distribution and enzymatic metabolism. The definition ofdrug latentiation has also been extended to include nonenzymaticregeneration of the parent compound. Regeneration takes place as aconsequence of hydrolytic, dissociative, and other reactions notnecessarily enzyme mediated. The terms prodrugs, latentiated drugs, andbioreversible derivatives are used interchangeably. By inference,latentiation implies a time lag element or time component involved inregenerating the bioactive parent molecule in vivo. The term prodrug isgeneral in that it includes latentiated drug derivatives as well asthose substances which are converted after administration to the actualsubstance which combines with receptors. The term prodrug is a genericterm for agents which undergo biotransformation prior to exhibitingtheir pharmacological actions.

[0042] As set out above, the present invention relates to a method fortreating a human with human immunodeficiency virus infection whichcomprises administering to the human a therapeutically effective amountof a thymidine analog, which analog acts as an inhibitor of viralreverse transcriptase necessary for viral replication of humanimmunodeficiency virus, and a thymidylate synthase inhibitor.

[0043] The thymidine analogs, and prodrugs thereof, which may beemployed in the present invention are compounds which act as inhibitorsof viral reverse transcriptase necessary for viral replication of humanimmunodeficiency virus. In general, the thymidine analogs are prodrugswhich are converted by cellular enzymes to their respective activemonophosphates, diphosphates, and/or triphosphates which are inhibitorsof viral reverse transcriptase. Nonlimiting examples of thymidineanalogs may be selected from the group consisting of3′-azido-3′-deoxythymidine, and D4T. In a preferred embodiment, thethymidine analog is 3′-azido-3′-deoxythymidine.

[0044] 3′-Azido-3′-deoxythymidine (AZT, azidothymidine, zidovudine,Retrovir™), is an antiretroviral drug active against humanimmunodeficiency virus. 3′-Azido-3′-deoxythymidine is an inhibitor ofthe replication of retroviruses including HIV also known as HTLV 111,LAV, or ARV. 3′-Azido-3′-deoxythymidine is a thymidine analog in whichthe 3′-hydroxy (—OH) group of thymidine is replaced by an azido (—N₃)group. Cellular thymidine kinase converts 3′-azido-3′-deoxythymidineinto AZT monophosphate. The monophosphate is further converted into AZTdiphosphate by cellular thymidylate kinase and to the AZT triphosphatederivative by other cellular enzymes. 3′-Azido-3′-deoxythymidinetriphosphate interferes with the HIV viral RNA dependent DNA polymerase(reverse transcriptase) and thus, inhibits viral replication.3′-Azido-3′-deoxythymidine is useful in treating humans Identified ashaving HIV infection. 3′-Azido-3′-deoxythymidine is disclosed in J. R.Horwitz et al., J. Org. Chem. 29, July 1964, pp. 2076-2078; M. Imazawaet al., J. Org. Chem., 43(15) 1978, pp. 3044-3048; also see BiochemicalPharmacology, Vol. 29, pp. 1849-1851; C. J. Kreig et al., ExperimentalCell Research 116 (1978) pp. 21-29; W. Ostertag et al, Proc. Nat. Acad.Sci. U.S.A. 71 (1974).

[0045] The thymidine analogs which act as an inhibitor of viral reversetranscriptase necessary for viral replication of human immunodeficiencyvirus, may be administered as the free base or in the form of apharmaceutically acceptable salt, e.g., an alkali metal salt such assodium or potassium, an alkaline earth salt or an ammonium salt (all ofwhich are hereinafter referred to as a pharmaceutically acceptable basesalt). The salts of the thymidine analog are converted to the free baseafter being administered to the human and are thus prodrugs.

[0046] The amount of thymidine analog which acts as an inhibitor ofviral reverse transcriptase present in the therapeutic compositions ofthe present invention is a therapeutically effective amount. Atherapeutically effective amount of thymidine analog is that amountnecessary to inhibit viral reverse transcriptase. All prodrugs orprecursors are administered to a human in a therapeutically effectiveamount sufficient to generate an effective amount of the compound whichinhibits viral reverse transcriptase necessary for viral replication ofhuman immunodeficiency virus. In general, a suitable effective dose ofthe thymidine analog or its pharmaceutically acceptable basic salts willbe in the range of about 5 mg to 250 mg per kilogram body weight ofrecipient per day, preferably in the range of 7.5 mg to 100 mg perkilogram body weight per day, and most preferably in the range 10 mg to40 mg per kilogram body weight per day.

[0047] The thymidylate synthase inhibitors, and prodrugs thereof, whichmay be employed in the present invention are compounds which areantimetabolites which interfere with the synthesis of deoxyribonucleicacid (DNA) and to a lesser extent inhibit the formation of ribonucleicacid (RNA). In general, the thymidylate synthase inhibitors inhibit thesynthesis of thymidine triphosphate so that the phosphorylated thymidineanalog which acts as an inhibitor of the viral reverse transcriptase cancompete more effectively with thymidine triphosphate and will moreeffectively inhibit viral reverse transcriptase necessary for viralreplication of human immunodeficiency virus. Nonlimiting examples ofthymidylate synthase inhibitors may be selected from the groupconsisting of 5-fluorouracil, 5-fluoro-2-pyrimidone (a prodrug of5-fluorouracil), and floxuridine. Preferably, the thymidylate synthaseinhibitor is floxuridine. These drugs may inhibit HIV infection by othermechanisms as well.

[0048] 5-Fluorouracil (5-FU) is a fluorinated pyrimidine antineoplasticantinetabolite. The metabolism of 5-fluorouracil in the anabolic pathwayblocks the methylation reaction of deoxyuridylic acid to thymidylic acidand interferes with the synthesis of deoxyribonucleic acid (DNA) and toa lesser extent inhibits the formation of ribonucleic. acid (RNA). SinceDNA and RNA are essential for cell division and growth, the effect offluorouracil may be to create a thymine deficiency which provokesunbalanced growth and death of the cell. The effects of DNA and RNAdeprivation are most marked on those cells which grow more rapidly andwhich take up fluorouracil at a more rapid pace.

[0049] Floxuridine (FUdr) is a fluorinated pyrimidine antineoplasticantimetabolite. Chemically, floxuridine is 2′-deoxy-5-fluorouridine.FUdr produces the same toxic and antimetabolic effects as does5-fluorouracil. The primary effect is to interfere with the synthesis ofdeoxyribonucleic acid (DNA) and to a lesser extent inhibit the formationof ribonucleic acid (RNA).

[0050] Derivatives of 5-fluorouracil and floxuridine may also beincorporated into DNA or RNA.

[0051] The amount of thymidylate synthase inhibitor present in thetherapeutic compositions of the present invention is a therapeuticallyeffective amount. A therapeutically effective amount of thymidylatesynthase inhibitor is that amount necessary to improve the antiviralefficacy of the thymidine analog so that the phosphorylated thymidineanalog which acts as an inhibitor of the viral reverse transcriptase cancompete more effectively in the inhibition of viral reversetranscriptase necessary for the replication of HIV. In general, asuitable effective dose of the thymidylate synthase inhibitor or itspharmaceutically acceptable salts will be in the range of about 0.01 mgto 25 mg per kilogram body weight of recipient per day, preferably inthe range of 0.01 mg to 10 mg per kilogram body weight per day, and mostpreferably in the range 0.01 mg to 5 mg per kilogram body weight perday.

[0052] As set out above, the method of the present invention may furthercomprise administering to a human a therapeutically effective amount ofa folate antagonist together with the thymidine analog which acts as aninhibitor of viral reverse transcriptase and the thymidylate synthaseinhibitor to modulate the effects of the thymidine analog. The folateantagonists, and prodrugs thereof, which may be employed in the presentinvention are compounds which are antimetabolites which interfere withthe synthesis of deoxyribonucleic acid (DNA) and to a lesser extentinhibit the formation of ribonucleic acid (RNA). Nonlimiting examples offolate antagonists may be selected from the group consisting ofmethotrexate and trimetraexate. Preferably, the folate antagonist ismethotrexate.

[0053] Methotrexate (Amethopterin) is an antimetabolite used in thetreatment of certain neoplastic diseases, severe psoriasis, and adultrheumatoid arthritis. Chemically methotrexate isN-[4-[[(2,4-diamino-6-pteridinyl)-methyl]methylamino]benzoyl]-L-glutamicacid. Methotrexate inhibits dihydrofolic acid reductase. Dihydrofolatesmust be reduced to tetrahydrofolates by this enzyme before they can beutilized as carriers of one carbon groups in the synthesis of purinenucleotides and thymidylate. Therefore, methotrexate interferes with DNAsynthesis, repair, and cellular replication.

[0054] The amount of folate antagonist present in the therapeuticcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of folate antagonist is thatamount necessary to modulate the effects of the thymidine analog. Ingeneral, a suitable effective dose of folate antagonist or itspharmaceutically acceptable salts will be in the range of about 0.05 mgto 25 mg per kilogram body weight of recipient per day, preferably inthe range of 0.05 mg to 10 mg per kilogram body weight per day, and mostpreferably in the range 0.05 mg to 4 mg per kilogram body weight perday.

[0055] As set out above, the method of the present invention may furthercomprise administering to a human a therapeutically effective amount ofhydroxyurea, and prodrugs thereof, together with the thymidine analogand the thymidylate synthase inhibitor to modulate the effects of thethymidylate synthase inhibitor. Hydroxyurea has the structural formulaH₂N—CO—NHOH. The precise mechanism by which hydroxyurea producescytotoxic effects is not known but it is believed that hydroxyureacauses an immediate inhibition of DNA synthesis without interfering withthe synthesis of ribonucleic acid or of protein.

[0056] The amount of hydroxyurea present in the therapeutic compositionsof the present invention is a therapeutically effective amount. Atherapeutically effective amount of hydroxyurea is that amount necessaryto modulate the effects of the thymidylate synthase inhibitor. Ingeneral, a suitable effective dose of hydroxyurea or itspharmaceutically acceptable salts will be in the range of about 5 mg to250 mg per kilogram body weight of recipient per day, preferably in therange of 7.5 mg to 100 mg per kilogram body weight per day, and mostpreferably in the range 10 mg to 40 mg per kilogram body weight per day.

[0057] Administration may be by any suitable route including oral,rectal, nasal, topical (including buccal and sublingual), vaginal, andparenteral (including subcutaneous, intramuscular, intravenous andintradermal), with oral or parenteral being preferred. The preferredroute may vary with the condition and age of the recipient.

[0058] While it is possible for the administered ingredients to beadministered alone, it is preferable to present them as part of apharmaceutical formulation. The formulations of the present inventioncomprise the administered ingredients, as above defined, together withone or more acceptable carriers thereof and optionally other therapeuticingredients. The carrier(s) must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient.

[0059] The formulations include those suitable for oral, rectal, nasal,topical (including buccal and sublingual), vaginal or parenteral(including subcutaneous, intramuscular, intravenous and intradermal)administration. The formulations may conveniently be presented in unitdosage form, e.g., tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

[0060] Such methods include the step of mixing the ingredients to beadministered with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

[0061] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension.

[0062] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surfactant or dispersingagent. Molded tablets may be made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active ingredienttherein.

[0063] Formulations suitable for topical administration include lozengescomprising the ingredients in a flavored base, usually sucrose andacacia or tragacanth; pastilles comprising the active ingredient in aninert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

[0064] Formulations suitable for topical administration to the skin maybe presented as ointments, creams, gels and pastes comprising theingredient to be administered and a pharmaceutically acceptable carrier.A preferred topical delivery system is a transdermal patch containingthe ingredient to be administered.

[0065] Formulations for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

[0066] Formulations suitable for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size, forexample, in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

[0067] Formulations suitable for vaginal administration may be presentedas pessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

[0068] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented in unitdose or multidose containers, for example, sealed ampules and vials, andmay be stored in a freeze-dried (lyophilized) condition requiring onlythe addition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

[0069] Preferred unit dosage formulations are those containing a dailydose or unit, daily subdose, or an appropriate fraction thereof, of theadministered ingredient.

[0070] The present invention is further illustrated by the followingexamples which are presented for purposes of demonstrating, but notlimiting, the preparation of the compounds and compositions of thisinvention.

EXAMPLES Sanctuary Growth of HIV in the Presence of AZT MethodsConstruction of Recombinant Proviral DNA

[0071] The HIV construct encoding LacZ has been described (26). Itcontains the LacZ gene driven by an SV40 promoter inserted into a largedeletion in the HIV genome extending from the 5′ end of the pol gene tothe 3′ end of the env gene. The HIV-gpt and HXB2env plasmids were kindlyprovided by Kathleen Page (University of California, San Francisco,Calif.) (18).

[0072] The HIV-gpt plasmid contains an HXB2 provirus into which an SV40promoter gpt (E. coli guanine phosphoribosyl transferase) gene wasinserted into the env region. The HXB2 env plasmid contains the HXB2gp160 gene driven by an SV40 promoter.

Production of “Plasmid Derived” Recombinant Retroviruses

[0073] All transfections and cell culture were performed in an approvedfacility using BSL3 techniques. Plasmid DNA co-transfections into COScells were performed as described by Page et al. (18). Supernatants fromCOS cells were collected 40 hours after transfection and assayed forinfectious recombinant HIV-LacZ virus by inoculating 2×10⁵ HeLa-T4 cellswith 0.1 ml of filtered (0.45 μm) supernatant. Cells were stained forbeta-galactosidase activity with X-gal 48 hours after infection asdescribed (26,27). To assay for infectious recombinant HIV-gpt virus,the infected cells were split 1:10 into gpt selective media as described(26). Medium changes were performed every 3 days and colonies werecounted 10-14 days post-infection after staining with 1% crystal violetin 10% formalin.

Cell Lines Containing Defective HIV-gpt and HIV-LacZ

[0074] The H9/HIV-gpt cell line and HeLa T4/HIV-LacZ cell line wereprepared and used as previously described (26). Rescue of defectiveretroviruses from the H9/HIV-gpt cell line and the HeLa T4/HIV-LacZ cellline were performed as previously described (26). Following each rescueinfection, the resultant titer of HIV-LacZ or IUV-gpt was determined andthe inoculum used to infect HeLa-T4 cells was adjusted depending uponthe number of infectious events to be analyzed.

HPLC Analysis of Clones

[0075] Cell lines were incubated with ³H-thymidine or ³H-AZT for 4hours. Dried methanol extracts of the clones were redissolved in 60 μlof distilled water and centrifuged to remove undissolved material.Twenty microliters of the sample was injected and separated on a 10×100mm Rainin Hydropore anion exchange column. The nucleosides were elutedfrom the column with a linear gradient of potassium phosphate (5 mM to 1M, pH 4.0) at a flow rate of 1 ml/min. The samples were collected (0.5ml), mixed with 5 ml Packard scintillation fluid, and quantitated usinga liquid scintillation counter. Phosphorylated derivatives of thymidineand AZT were identified with authentic standards.

Cytotoxicity Assay

[0076] AZT-mediated cytotoxicity was assayed in cells persistentlyrefractory to the antiviral effects of AZT (R116) and in cells sensitiveto the antiviral effects of AZT (HT4, S pool and S1) using a standardassay (14). Triplicate wells of 24—well plates containing 3×10⁴ cellswere cultured in the absence or presence of various concentrations ofAZT. Three days later, drug cytotoxicity was quantitated with a standardMTT assay in which the uptake and metabolism of3-[4,5-dimethylthiazol-2-yl] 2,5-dephenyltetrazolium bromide (MTT) bycells was measured (14). The amount of formazan produced in 2 hours wasdetermined by dissolving the product in 100% DMSO and then measuring theabsorbance at 570 nm.

Northern Blot Analysis

[0077] Total RNA from S1 and R116 cells were extracted as describedpreviously (4). Equal amounts of total RNAs were electrophoresed on anagarose gel containing 1% formaldehyde and blotted onto a nylonmembrane. The RNAs were hybridized with a ³²P-labled human thymidinekinase probe (3). The labeled bands were visualized usingautoradiography and quantitated using a Molecular Dynamics PersonalDensitometer.

Thylnidine Kinase Assay

[0078] Thymidine kinase activity was determined in cell lines sensitiveand resistant to AZT. Cellular extracts of S1 and R116 cells wereprepared according to Sherley and Kelly (24) and assayed for thymidinekinase activity as described by Lee and Cheng (10). Proteinconcentration of each extract was determined using Biorad ProteinReagent.

Use of Floxuridine to Modulate the Antiviral Activity of AZT Materialsand Methods

[0079] Cells. Cell line R116 is a derivative of HeLa-T4 cells that wasisolated after infection of HeLa-T4 cells with HIV-gpt in the presenceof 10 μM AZT (31). This cell line was demonstrated to be refractory tothe antiviral effects of AZT by virtue of reinfection with eitherrecombinant or replication-competent HIV infection in the presence ofAZT. Cell line S1 is a derivative of HeLa-T4 cells that was isolatedafter infection of HeLa-T4 cells with HIV-gpt in the absence of AZT(31). Cells were cultured in Dulbecco's modified Eagle's mediumsupplemented with antibiotics, 2 mM L-glutamine, and 10% fetal bovineserum (FBS). H9 cells, JE6.1 cells and MT-2 cells were cultured in RPMI1640 medium supplemented with antibiotics, 2 mM L-glutamine, and 10%FBS. Peripheral blood mononuclear cells (PBMC) isolated from healthyHIV-1 seronegative donors were activated with PHA (10 ug/ml) for 72hours prior to HIV-1 infection. PBMC were maintained in RPMI 1640supplemented with 10% interleukin-2 (Advanced Biotechnologies, Columbia,Md.), 20% FBS, 2 mM L-glutamine and antibiotics.

Virus

[0080] Stock preparations of HIV-1 IIIB were harvested from H9 cells bythe “shake off method” (13). An AZT sensitive clinical isolate(HIV-1_(preAO8)) (9) was prepared in MT-2 cells. Stock virus infectivitywas determined by end-point dilution in MT-2 cells (32). Virus-inducedcytopathic effect (syncytium formation) was scored 7 days post-infectionand the TCID₅₀ was calculated with the Reed and Muench equation (33).

Compounds

[0081] Azidothymidine (AZT) and Floxuridine (FUDR) were purchased fromSigma Chemical Co. (St. Louis, Mo.) and were dissolved in phosphatebuffered saline, sterile filtered (0.22 um) and stored at −20° C.

HIV RT Assay

[0082] HIV-1 production in infected cultures was determined by a³²P-based assay as described (34). RT activity was determined byqualification of ³²P bound to the DE81 paper by using a MolecularDynamics phosphorimager. The results are reported as pixel units permicroliter of the reaction mixture.

Cytotoxicity Assay

[0083] A checkerboard analysis of the cytotoxicity of AZT and FUDR aloneand in combination was assayed. Triplicate wells of of 24-well platescontaining 1×10⁵ cells were cultured in the absence or presence ofvarious concentrations of each drug alone and in combination. Sampleswere taken every two days for 8-10 days. Drug cytotoxicity wasquantitated by the MTT reduction assay (14). The amount of formazanproduced in 4 hours was determined by dissolving the product in 0.1N HClmade with 2-propanol and then measuring the A₅₇₀.

Early HIV Breakthrough Infection in the Presence of Stavudine Materialsand Methods

[0084] Cells: The lymphoid cell lines H9 and JE6.1 were cultured in RPMI1640 medium supplemented with antibiotics, 2 mM L-glutamine and 10% FBS.Peripheral blood mononuclear cells (PBMC) isolated from healthy HIV-1seronegative donors were activated with PHA (10 ug/ml) for 72 hoursprior to infection. After PHA stimulation, PBMCs were maintained in RPMI1640 supplemented with 10% interleukin-2 (Advanced Biotechnologies,Columbus, Md.), 20% FBS, 2 mM L-glutamine and antibiotics.

[0085] Virus: Production of recombinant HIV-gpt has been describedelsewhere (26). The amphotropic cell line PA317 was transfected with therecombinant murine retrovirus pLXSN (36) and was used as the source ofthe recombinant MLV-neo virus. Stock preparations of HIV-1IIIB wereharvested from H9 cells by the “shake off method” (13). Stock virusinfectivity was determined by end-point dilution in MT-2 cells (32).Virus induced cytopathic effect was scored 7 days post-infection and theTCID50 was calculated with the Reed and Muench equation (33).

[0086] Compounds: Stavudine (D4T) and Floxuridine (FUdr) were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.) and were dissolved in phosphatebuffered saline, sterile filtered and stored at −20° C.

[0087] HIV-1 RT assay: HIV-1 production in infected cells was determinedby a ³²P-based assay as described (37). RT activity was determined byquantification of ³²P-bound to DE81 paper by using a Molecular Dynamicsphosphorimager. The results are reported as pixel units per microliterof the reaction mixture.

[0088] Cytotoxicity assay: A checkerboard analysis of the cytotoxicityof D4T and FUdr alone and in combination was assayed. Triplicate wellsof 24 well plates containing 1×105 cells were cultured in the absence orpresence of various concentrations of each drug alone or in combination.Samples were taken every two days for 8-10 days. Drug cytotoxicity wasquantitated by the MTT reduction assay (14). The amount of formazanproduced in 4 hours was determined by dissolving the product in 0.1N HCLmade with 2-propanol and the measuring the A570.

Results Sanctuary Growth of HIV in the Presence of AZT HIV-gpt Infectionof Cells in the Absence and Presence of AZT

[0089] HeLa-T4 cells were infected with a recombinant HIV, HIV-gpt, inthe presence or absence of 10 μM AZT (FIG. 1). Two separate populationsof HIV-gpt were utilized for these infections. One population of HIV-gptwas produced in COS cells by co-transfection of the HIV-gpt plasmid witha plasmid encoding the HXB2 env gene. The infectious virions produced bythis co-transfection have little genetic diversity in that they areproduced from products encoded by plasmids in COS cells. The secondpopulation of HIV-gpt was genetically more diverse, being produced byrescue from the H9/HIV-gpt cell line with replication competentHIV-1IIIB that had been propagated in culture (26). After infection, theHeLa-T4 cells were placed in gpt selective media and the number ofcolonies developing by day 10 was used as an indicator of the number ofcells initially infected in the absence or presence of 10 μM AZT. As canbe seen in Table 1, the prevalence of colony formation after infectionin the presence of AZT was similar (−5×10⁴) with the two preparations ofHIV-gpt. This similarity is very distinct from the results of infectionsperformed in the presence of a normucleoside reverse transcriptaseinhibitor, TIBO R82150. In those studies, the prevalence of infectionwith the COS-cell derived virus was twenty fold lower than infectionwith HIV-gpt rescued by replication-competent virus (26). Since theHIV-gpt produced in COS cells would not be expected to be geneticallydiverse, this relatively high rate of infection in the presence of AZTwas not likely due to the detection of viral encoded-AZT resistance.Similarly, the absence of more prevalent infection in the presence ofAZT when HIV-gpt was produced by rescue with a propagated stock ofreplication-competent HIV, implies that true genetic resistance was notbeing detected in these experiments. These data suggest that othermechanisms may contribute to this early viral breakthrough in thepresence of AZT.

Identification of Cells Refractory to the Antiviral Effects ofNucleoside Analogs

[0090] To characterize further the mechanism(s) of viral infectionaccounting for the high frequency of colony formation after infection inthe presence of 10 μM AZT, the experiment depicted in FIG. 2 wasperformed. HeLa-T4 cells were infected with HIV-gpt (prepared in COScells) in the absence or presence of AZT. Infected cells were selectedin gpt selective media and colonies were isolated and expanded into celllines. Twelve cell lines developing after infection in the presence ofAZT were further characterized. To determine if these cell lines wererefractory to the antiretroviral effects of AZT they were infected withHIV-LacZ in the presence of 10 μM AZT. Three days after infection, thecells were stained with X-gal to detect 13-galactosidase activity. Nineof these twelve cell lines behaved like wild type HeLa-T4 cells withcomplete inhibition of infection in the presence of AZT. However, threeof these cell lines demonstrated greater than 50% of control infection(-AZT) despite the presence of 10 μM AZT. These cell lines were labeledas “persistently resistant” to the antiretroviral effects of AZT.

[0091] Infection of these “persistently resistant” cell lines withreplication-competent HIV confirmed the relative inefficacy of AZT inthese cells. For example, a clinically relevant concentration of 0.1 μMAZT was much less effective in inhibiting HIV-1IIIB in the “persistentlyresistant” cell line than in the control cells (FIG. 3). No such cellsresistant to the antiviral effects of AZT were obtained when coloniesderived from HIV-gpt infections in the absence of AZT were studied (seeTable 2).

[0092] None of the “persistently resistant” cell lines were cross“resistant” to the antiretroviral effects of ddl or ddC. Interestingly,cells with persistent resistance to AZT showed partial cross resistanceto the antiretroviral effects of 50 μM d4T. In addition to thisevaluation for cellular cross-resistance, it was possible to use asimilar experimental protocol to demonstrate the independent selectionof cells refractory to the antiretroviral effects of a variety of othernucleoside analogs (Table 2). In contrast, no cells “persistentlyresistant” to the antiretroviral effects of the non-nucleoside reversetranscriptase inhibitor TIBO R82150 could be selected using identicaltechniques. These results indicate that HeLa-T4 cells havesubpopulations of cells that are independently refractory to theantiretroviral effects of a variety of nucleoside analogs.

Comparison of Thymidine and AZT Phosphorylation in Isolated Clones

[0093] To initiate an analysis of the mechanisms responsible for thiscellular resistance, a persistently resistant cell line was compared toa control cell line obtained by HIV-gpt infection in the absence of AZT.Each of these cell lines was incubated with ³H-thymidine and thymidinemetabolites were assayed by HPLC. As shown in FIGS. 4A and 4B, thepersistently resistant cell line (R116) had a greater phosphorylation ofthymidine into TTP compared to the non-resistant cell line (SI). Anidentical experiment with ³H-AZT indicated a nearly 2 fold reduction inAZTTP in R116 cells compared to Si cells (Table 3). Therefore, acomponent of the resistance may be related to a diminished AZTTP/TTPratio. These results suggest that alterations in nucleotide metabolismmay underlie some of the differences between these cell lines. Tofurther characterize the basis for these differences, thymidine kinasemRNA levels and thymidine kinase activity were compared in the two celllines. Although there were no differences in the thymidine kinase mRNAlevels on a Northern blot analysis, the R116 cell line had 3 timesgreater thymidine kinase activity than the S1 cell line (FIG. 5).

Tolerance of the Clones to Very High Concentrations of AZT

[0094] In additional studies of these cell lines, tolerance of highconcentrations of AZT was tested. As shown in FIG. 6, the persistentlyresistant cell line (R116) was much more tolerant of high concentrationsof AZT. The cytotoxic concentration of AZT that killed 50% of a varietyof control cell lines was approximately 100 μM. In contrast, thecytotoxic concentration of AZT that killed 50% of the persistentlyresistant clone R116 was greater than 1 mM. This implies that themechanisms that protect HIV from AZT in the resistant cell lines alsoprotect these cell lines from the cytotoxic effects of even higherconcentrations of AZT. This demonstrates another AZT-related differenceamongst these clones derived from the same parental cell line.

Use of Floxuridine to Modulate the Antiviral Activity of AZT Inhibitionof Early Viral Breakthrough in Cells Sensitive and Refractory to theAntiretroviral Effects of AZT

[0095] A previous study has utilized replication-defective HIV toquantitate early infection in the presence of AZT (31). In that study,HIV-gpt (a recombinant HIV encoding a selectable marker) was used toinfect HeLa-T4 cells in the presence of AZT. Infected cells wereisolated in gpt selective media and expanded into cell lines. Severalsuch cell lines were refractory to the antiviral effects of AZT asevidenced by the ability of replication-defective orreplication-competent HIV to infect these cells in the presence of AZT.Several control cell lines were obtained by infection of HeLa-T4 cellswith HIV-gpt in the absence of AZT. Cell line R116 is a cell line thatwas determined to be refractory to the antiviral effects of AZT. Cellline SI is a control cell line. A prior metabolic analysis of these celllines indicated that cell line R116 had a reduced accumulation of AZTTPand an increased phosphorylation of thymidine to TTP in comparison tothe SI control cell line (31). To determine if the addition of afluoropyrimidine to AZT increased the antiviral efficacy of AZT in theR116 cell line, cells were cultured in the absence or presence of 0.1 μMAZT or 0.01 μM FUdR alone or in combination prior to infection withHIV-1 IIIB at an input multiplicity of infection of 1. As demonstratedin FIG. 7A, 0.1 μM AZT had potent antiviral efficacy in the control S1cell line. In contrast, in the cell line refractory to the antiviraleffects of AZT (R116) there was significant HIV replication in thepresence of 0.1 μM AZT (FIG. 7B). However, the addition of 0.01M FUdR to0.1 μM AZT suppressed this viral breakthrough. At these concentrations,no cytotoxicity was observed. To further characterize these differentcell lines, cytotoxicity to various concentrations of FUdR weredetermined. As shown in FIG. 8, the R116 cell line had an ED₅₀ of 0.7 μMFUdR whereas the S1 cell line, parental HeLa-T4 cells and a pool ofcontrol cell lines all had an ED50 of 7 μM. These results furthersubstantiate the presence of metabolic differences in cells refractoryto the antiviral effects of AZT as opposed to cells sensitive to theantiviral effects of AZT.

Efficacy of AZT in Combination with FUdR in Inhibiting HIV-1 Infectionof Lymphoid cells Sensitive and Refractory to the Antiviral Activity ofAZT

[0096] To extend the analysis of the antiviral efficacy of AZT incombination with FUdR to lymphoid cells, similar experiments wereperformed in Jurkat JE6.1 cells. In these experiments, three populationsof cells were studied. The parental JE6.1 cells were compared topopulations of JE6.1 cells that were isolated after infection with areplication-defective recombinant Moloney Leukemia virus containing theTn5 neo gene (MLV-neo) in the absence or presence of AZT. JE6.1 cellswere infected in the absence or presence of 10 μM AZT and two days laterthe cells were placed in media containing 1 mg/ml G418 to allow thegrowth of infected cells. JE6.1AZTR is the cell population that wasinfected with MLV-neo in the presence of AZT. JE6.1con is the controlpopulation of cells infected with MLV-neo in the absence of AZT.

[0097] The efficacy of AZT in combination with FUdR in inhibiting HIV-1infection of these cell populations was determined by infection in theabsence of AZT or in the presence of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM or10 μM AZT in combination with no FUdR, 0.005 μM FUdR, 0.01 μM FUDR or0.025 μM FUdR. This experiment allowed a detailed analysis of the IC₅₀of AZT in each population with different concentrations of FUdR. Theseresults are shown in Table 4. Based upon prior results in HeLa-T4 cells(31), it is likely that the JE6.1AZTR cells represent a mixture ofcells, some of which require an increased concentration of AZT toinhibit HIV infection. This is reflected by a 2 fold increase AZT IC₅₀when analyzing the entire population.

[0098] Strikingly, the combination of FUdR with AZT dramaticallysuppresses HIV infection of this population. A greater than 600 foldreduction of AZT IC₅₀ is seen during infection of these cells in thepresence of AZT and FUdR. In fact, these cells, which were initiallyisolated as cells infected in the presence of AZT, were more sensitiveto the antiviral effects of the AZT-FUdR combination than were controlor parental cells. These results suggest that this population of cellshas metabolic features that renders them highly susceptable to theantiviral effects of the AZT-FUdR combination. Of note, there is a10-fold reduction of AZT IC₅₀ when the parental and control cellpopulations were infected with HIV-1 in the presence of AZT and FUdR. Nocytotoxicity was observed in the AZT-FUdR combination except at thehighest drug concentrations used (10 μM AZT plus 0.025 μM FUdR, FIG. 9).

Efficacy of AZT in Combination with FUdR in Inhibiting HIV-1 Infectionof Primary Blood Mononuclear Cells

[0099] The antiviral efficacy of the combination AZT and FUdR was alsoassessed in PBMC. These studies are shown in FIG. 10 and demonstratethat the combination of AZT and FUdR has potent antiviral activity inPBMC. Similar results were obtained with a primary HIV isolate known tobe genetically sensitive to AZT. Therefore, FUdR potentiates theantiviral efficacy of AZT in PBMC infected with either HIVIIIB or aclinical isolate. Of note, cytotoxicity in the AZT-FUdR combination wassimilar to that seen for the JE6.1 cells in that cytotoxicity was onlyobserved when 1 0 μM AZT was combined with 0.025 μM FUDR.

Early HIV Breakthrough Infection in the Presence of StavudinePreliminary Characterization of Mechanisms Allowing HIV Infection in thePresence of d4T

[0100] To undertake a preliminary characterization of the predominantmechanisms responsible for early HIV infection after the initiation ofd4T, we utilized several populations of recombinant viruses. Recombinantreplication-defective HIV was prepared by transfection of COS cells withcomplementing plasmids encoding the RNA and proteins necessary for theproduction of a recombinant HIV encoding gpt (26). Such viruses areproduced without major genetic heterogeneity as the predominantmechanisms responsible for the generation of heterogeneity (e.g., cyclesof reverse transcription) are not involved in the production of theseviruses. Even if there was heterogeneity as a consequence of errorsduring plasmid transcription, the resulting mutated proteins would begreatly diluted in a population of proteins. In contrast, replicationdefective HIV-gpt made by rescue with replication-competent HIV willcontain proteins encoded by the replication-competent virus used forrescue. Prior experiments have demonstrated the close relationshipbetween the drug sensitivity phenotype of the recombinant virus and thedrug sensitivity phenotype of the virus used to rescue the recombinantvirus (26). Therefore, recombinant virus produced by rescue withreplication-competent virus will be heterogeneous and may reflect thedrug sensitivity profile of the virus used for rescue. In a priorexperiment, the heterogeneity introduced by the replication-competentvirus used for rescue resulted in a calculation of the prevalence of HIVresistant to a NNRTI in an unselected population (26). This calculatedvalue was very similar to the prevalence subsequently calculated from invivo studies of HIV dynamics after the initiation of a NNRTI (38).

[0101] A comparison of the two virus populations described abovedemonstrated very similar rates of infection in the presence of highconcentrations of d4T (Table 5). This high level of infection with avirus produced from plasmid transcripts (HIV-gpt) suggested a high rateof infection with a homogenous population of a recombinant virus whoseproteins were generated by translation of plasmid transcripts and thusnot anticipated to have a high level of genetic heterogeneity. Toconfirm this high rate of infection in the absence of genetic drugresistance, we used several other recombinant viruses and host cells.MLV based recombinant viruses showed a similar high rate of infection inthe presence of d4T (Table 6). Similar infections in the presence ofhigh concentrations of other nucleoside reverse transcriptase inhibitorshave consistently demonstrated a rate of infection in the presence ofhigh concentrations of d4T greater than that seen in the presence ofhigh concentrations of the other nucleoside analogs (Table 6). A highrate of infection of Jurkat cells in the presence of d4T has also beenseen.

[0102] The similar rates of infection with virus prepared by plasmidtransfection and virus prepared by rescue with replication-competent HIVsuggested that genetic resistance was not the major mechanism of earlyHIV breakthrough being detected. This interpretation was supported byevidence of high rates of infection with MLV based recombinant virions(also anticipated to have a low level of genetic heterogeneity).Infections of Jurkat cells indicated that the high rate of infection inthe presence of high concentrations of d4T was not cell line specific.These data suggested that early HIV breakthrough infection in thepresence of d4T was not due to infection by d4T-resistant virus.

Isolation of the Cells Infected with HIV in the Presence of d4T

[0103] The presence of a selectable marker gene in the recombinant HIVallowed the isolation of cells infected by the recombinant viruses inthe presence of d4T. These cells were characterized by infection withboth additional recombinant viruses and by replication-competentviruses. As demonstrated in Table 6, approximately 37% of the isolatedcells were repeatedly refractory to the antiviral effects of d4T (i.e.,they could be readily re-infected with recombinant HIV, recombinant MLV,or replication-competent HIV in the presence of high concentrations ofd4T). An even higher percentage of the Jurkat cells infected in thepresence of d4T were persistently refractory to the antiviral effects ofd4T (Table 7). Infections with replication-competent HIV demonstratedthat the refractoriness to infection detected in these clones was not aphenomena solely associated with recombinant viruses (FIG. 11). A subsetof these persistently refractory cells (approximately 20%) were alsorefractory to the antiviral effects of AZT.

Combined d4T-FUdR Antiviral Activity

[0104] As has been demonstrated previously for AZT, a component of therefractoriness to the antiviral effects of d4T can be reversed by theaddition of FUdR (Table 8). The antiviral efficacy of the combinationtherapy, as measured by the d4T IC50, is markedly improved withcombination therapy. Prior studies of the antiviral efficacy of FUdRhave demonstrated limited antiviral efficacy of FUDR alone, but markedantiviral efficacy of combined AZT and FUdR. Table 8 shows the capacityof the FUdR-d4T combination to reverse some of the cellularrefractoriness to d4T described above. The FUdR-d4T combination hasmarked antiviral activity in cells demonstrated to be refractory to theantiviral effects of d4T. The antiviral efficacy of the combination hasalso been studied in unselected PBMC (FIG. 12).

Discussion Sanctuary Growth of HIV in the Presence of AZT

[0105] The studies described above indicate that sanctuary growth of HIVmay occur in the presence of AZT and that early in treatment cellularresistance may make a large contribution to viral breakthrough. In fact,there was no quantitative difference in HIV breakthrough when HIV-gptprepared by transfection in COS cells was compared to HIV-gpt producedby rescue with replication-competent HIV. This suggests that a largepart of early infection in the presence of AZT may be a consequence ofcellular effects. At least two types of such sanctuary growth weredetected. Nine of the twelve cell lines analyzed did not have persistentresistance to the antiviral effects of AZT and may have had epigeneticalterations such as those that might occur at specific points in thecell cycle. In contrast, three of the twelve cell lines had persistentresistance to the antiviral effects of AZT, with both recombinant andreplication-competent HIV. In studies with replication-competent HIV,virtually complete inhibition of the infection of control cells wasobtained with a concentration of AZT that only reduced viral productionin a persistently resistant clone by 50%.

[0106] These cell lines refractory to the antiviral effects of AZT arelikely to have specific alterations that render AZT less effective.Metabolic studies suggest that some of this resistance may be due todifferences in nucleotide metabolism resulting in a reduction of AZTTPin the resistant cells. It will be important to further characterize anddefine the mechanisms responsible for cellular resistance becausereversal of this resistance may greatly reduce viral burden and delaythe outgrowth of virus with genetic resistance. It is important toemphasize that the cells that were detected as refractory to theantiviral effects of AZT were only exposed to AZT for a short period oftime. There was no preselection of cells prior to infection with therecombinant viruses.

[0107] Recent reports on nucleotide pool sizes in resting as opposed tostimulated blood mononuclear cells and different cell lines derived fromdifferent blood cell lineages have demonstrated marked differences thatmight translate into variable efficacies of nucleoside analogs withinpopulations of blood cells (6,15). Furthermore, other investigators havegrown cells in high concentrations of AZT for prolonged periods of timeand demonstrated the selection of cells with reduced levels of thymidinekinase activity (17). Additional data about metabolic differencesoccuring in the lymphocytes of patients treated with prolonged coursesof AZT also suggests that cellular resistance may contribute to HIVbreakthrough (1). Thus, cellular resistance is likely to contribute toviral breakthrough during an in vivo infection and multiple mechanismsmay contribute to cellular resistance. The prevalence of resistant cellsdetected in single cell lines derived during infection in these studiesraises interesting speculation concerning the prevalence of similarresistant cells during an in vivo infection involving multiple celltypes.

[0108] Earlier studies with recombinant viruses indicated that there isa high prevalence of genetically TIBO resistant HIV in an unselected HIVpopulation. As a consequence of this high prevalence and the lack ofcellular metabolism for TIBO, genetically resistant virus is rapidlyselected in vivo and in vitro. In contrast, AZT is metabolized in cells,a subpopulation of which is refractory to the antiretroviral effects ofAZT. Early growth of “non-genetically resistant” virus can occur inthese sanctuary cells (“cellular resistance”). With continued growththere is amplification of pre-existing (or emerging) viral variants withgenetic resistance because the truly resistant virus can infect anysuitable target cell, not just those cells in which AZT is ineffective.This gives a relative growth advantage to the genetically resistantvirus. Subsequent additional mutations or recombination events mayresult in viruses with multiple mutations. The initial “cellularresistance” may allow a population of non-resistant or partiallyresistant virus to replicate, providing a pool of virus in whichadditional mutations and recombination events can occur. Reversal ofcellular resistance could conceivably delay, or even prevent, theoutgrowth of highly resistant virus with multiple mutations by notallowing non-resistant or partially resistant virus (with singlemutations) to replicate.

[0109]FIG. 1 is a schematic representation of the production ofrecombinant HIV-gpt by COS cell transfection or rescue from theH9/HIV-gpt cell line.

[0110]FIG. 2 is a schematic representation of the analysis of coloniesarising after COS cell derived HIV-gpt infection of HeLa-T4 cells in thepresence of 10 μM AZT. Twelve such colonies were expanded and infectedwith HIV-LacZ in the presence and absence of 10M AZT. Ten controlcolonies derived from HIVgpt infection of HeLa-T4 cells in the absenceof AZT were studied in parallel. “Persistent” cellular resistance wasdefined by a high level infection with HIVLacZ in the presence of AZT,as shown for colony number 2. HIV-LacZ contains the LacZ gene driven byan SV40 promoter inserted into a large deletion in the HIV genomeextending from the pol gene to the 3′ end of the env gene. HIVLacZ virusproduction has been previously described (16).

[0111]FIG. 3 is a graph showing the infection of a clone of HeLa-T4cells “persistently resistant” to the antiviral effects of AZT (cloneR116) and a control clone (S1) with replicationcompetent HIV-1IIIB inthe presence of 0.1 μM AZT. P24 was assayed, compared to a controlinfection in the absence of AZT and plotted as a function of time. P24values in the absence of AZT were 1857+104 ng/ml for S1 and 1717+113ng/ml for R116.

[0112]FIGS. 4A and 4B are graphs illustrating thymidine metabolism-HPLCanalysis of clones obtained after infection of HeLa-T4 cells withHIV-gpt in the presence and absence of AZT. FIG. 4A illustrates the SIcell line derived from HeLa-T4 cells after infection with HIV-gpt in theabsence of AZT. FIG. 4B illustrates the R116 cell line, which waspersistently resistant to the antiviral effects of AZT. The earliestpeak represents thymidine and the subsequent peaks represent TMP, TDP,and TTP.

[0113]FIG. 5 is a graph showing a comparison of thymidine kinase mRNAlevels (A) and enzyme activity (B) in cell lines sensitive andpersistently resistant to the antiretroviral effects of AZT. The mRNAlevels of S1 and R116 were 8390 and 8500 densitometry units,respectively. Thymidine kinase activity was based upon three independantexperiments performed in triplicate.

[0114]FIG. 6 is a graph showing cellular toxicity of AZT. The cell lineswere grown in the presence of the indicated concentrations of AZT.Cellular toxicity was then determined in cells persistently refractoryto the antiviral effects of AZT (R116) and in cells sensitive to theantiviral effects of AZT (HT4, S pool and S1) using a standard MTTassay. S pool was a pool of colonies derived from HIV-gpt infection ofHeLa-T4 cells in the absence of AZT. HeLa-T4 is the parental cell line.

[0115] Table 1 shows the frequency of HIV-gpt colony formation in thepresence and absence of AZT. Table 1 also shows a comparison of HIV-gptproduced in COS cells by transfection with plasmids and HIV-gpt producedby rescue from the H9/HIV-gpt-cell line after infection with HIV-1lilB(see FIG. 1). TABLE 1 Number of colonies Source of HIV-gpt −AZT +AZTFrequency Plasmid-derived (COS cells) 3.1 × 10⁴ 16 5.2 × 10⁻⁴ Rescuewith HIV-1 1.8 × 10⁴ 9 5.0 × 10⁻⁴

[0116] Table 2 shows colony formation and “persistent resistance” afterHeLa-T4 infection with plasmid derived HIV-gpt (produced in COS cells)in the presence of high doses of the indicated antiretroviral agents.

[0117] Concentrations of the antiretroviral agents were: AZT-10 μM,DDI-50 μM, D4T-50 μM and DDC-10 μM. TABLE 2 Number of Number of“persistently resistance” Drug colonies colonies Frequency No Drug(Control) 8800 0/10 0 AZT 12 3/12 3.4 × 10⁻⁴ D4T 50 not done — DDI 162/16 2.3 × 10⁻⁴ TIBO 3 0/3  0

[0118] Table 3 shows the concentration of phosphorylated AZT metabolitesin the “persistently resistant” (R116) and sensitive (SI) cell lines.Pool sizes were determined by incubation of cells with ³H-AZT for 4hours followed by cellular extraction and HPLC. The numbers areexpressed as pmoles/10⁶ cells. The numbers in parentheses represent thepercentage of total radioactive species in that pool. TABLE 3 Clone AZTAZTMP AZTDP AZTTP S1 0.0206(12.2) 0.1212(71.6) 0.0116(6.9) 0.0158(9.3)R116 0.0155(7.7) 0.1575(78.6) 0.0193(9.6) 0.0083(4.2)

Use of Floxuridine to Modulate the Antiviral Activity of AZT

[0119] Preliminary studies from our laboratory have demonstrated thatearly HIV infection of various cell lines in the presence of AZT is notthe consequence of infection with AZT-resistant virus. In both HeLa-T4cells and a lymphoid cell line (Jurkat JE6.1), the predominant componentof early HIV infection in the presence of AZT is a consequence ofinfection with AZT-sensitive virus (31). Clinical studies alsodemonstrate that early HIV infection in the presence of AZT occurs withAZT-sensitive virus (29). To characterize the mechanisms allowing thereplication of AZT-sensitive HIV in the presence of AZT, a metabolicanalysis of some of the cells infected with HUV in the presence of AZTin vitro was previously undertaken (31). Those studies demonstrated thata component of early infection with drug-sensitive virus was occuring ina subpopulation of cells with features that would be anticipated todecrease the antiviral efficacy of AZT. These studies were importantbecause they indicated that the reversal of early HIV infection in thepresence of AZT required interventions directed at features other thanviral drug-resistance. Based upon a prior study demonstrating increasedphosphorylation of thymidine to TTP and decreased AZTTP in a subset ofcells infected with drug-sensitive HIV in the presence of AZT,applicants have attempted to modulate the antiviral efficacy of AZT bycombining AZT therapy with floxuridine. These initial studies havedemonstrated the suppression of early viral breakthrough infection inthe presence of AZT with drug combinations that are readily achievablein vivo and are non-cytotoxic. In addition, there is a clearconcentration-response relationship when FUdR is added to AZT.

[0120] In addition to the determination that the AZT-FUdR combinationsuppressed HIV infection of cells that were infected with HIV in thepresence of AZT, the combination was much more effective than AZT aloneat inhibiting HIV infection of an unfractionated lymphoid cell line andPBMC. This increased efficacy was also demonstrated with a clinicalisolate. Therefore, the enhanced antiviral activity of the combinationtherapy is not restricted to cell lines, recombinant viruses, orlaboratory strains of virus and may therefore have clinical utility.

[0121] The increased efficacy of AZT-FUdR in suppressing HIV infectionof cells readily infected with HIV in the presence of AZT isparticularly striking. Since this population of cells is a mixture ofcells with and without persistent refractoriness to the antiviraleffects of AZT (i.e., infection of a subset of this population isrepeatedly refractory to the antiviral effects of AZT), the AZT IC₅₀ forthis population is only minimally elevated. Nevertheless, infection ofthis entire population is extremely sensitive to inhibition by theAZT-FUDR combination. The supersensitivity of infection of thispopulation of cells to combination therapy was unanticipated and islikely to be explained by metabolic features that are responsible forthe efficacy of the combination. Determination of the mechanismsresponsible for this supersensitivity to combined AZT-FUdR therapy mustawait metabolic analysis of thymidine, AZT and FUdR phosphorylatedintermediates in populations of cells and individual clones. It isimportant to note that in all of these studies FUdR has moderateantiviral activity when used by itself. The mechanisms by which thisinhibition occurs are also currently unknown and may also be related toperturbations of normal thymidine metabolite pools, direct inhibition ofviral or cellular processes or by incorporation into the viral DNAduring reverse transcription.

[0122] It is very likely that the long term ability of HIV to replicatein the presence of AZT is a consequence of the emergence ofAZT-resistant virus. Multiple mutations in RT are necessary for thedevelopment of this genetic AZT-resistance and these mutations emergeover several months-years. Suppression of early HIV replication withAZTsensitive virus in the presence of AZT could delay, or even preventthe emergence of AZT resistant virus by diminishing the substrate forsubsequent genetic changes. Therefore, studies that define themechanisms of early viral breakthrough infection have potential longterm therapeutic implications.

[0123] The clinical feasibility of combined fluoropyrimidine-AZT therapyneeds to be evaluated. At low concentrations the fluoropyrimidines areoften well tolerated by oncology patients with few significantneurologic, gastrointestinal or hematologic toxicities. The in vivo dosenecessary to improve the antiviral efficacy of AZT will need to bedetermined, however extrapolation from in vitro studies indicates thatcytotoxic concentrations of fluoropyrimidines will not be needed. PhaseI clinical studies of FUdR combined with AZT in patients with HIV-1infection will provide information about the feasability of combinationtherapy. In addition, other drugs with the ability to decrease Tm levelswill also be evaluated in pre-clinical studies.

[0124]FIG. 7 is a graph showing the suppression of viral breakthrough incells sensitive and refractory to the antiviral effects of AZT. Cellssensitive (S1) and refractory (R116) to the antiretroviral effects ofAZT were infected with HIV-1 IIIB in the absence of drug (open squares),0.1 μM AZT (solid squares), 0.01 μM FUdR (solid triangle) or acombination of 0.1 μM AZT plus 0.01 μM FudR (open triangle). Cell freesupernatants were assayed for RT activity every two days. Results arethe mean of triplicate cultures. Standard deviations were <15%.

[0125]FIG. 8 is a graph illustrating FUDR cytotoxicity in cellssensitive and refractory to the antiretroviral activity of AZT. Cellssensitive, parental HT4 (open circle), S1 (solid square), Spool (solidcircle) and refractory, R116 (open square) were grown in the presence ofvarious concentrations of FUdR. Three days latter, cell viability wasdetermined by the MTT reduction method. Spool cells are a population ofcontrol cells obtained by infection with HIV-gpt in the absence of AZT(7).

[0126]FIG. 9 is a graph showing AZT-FUdR cytotoxicity in JE6.1 cellssensitive and resistant to the antiviral effects of AZT. Cytotoxicity of10 μM AZT in combination with 0.025 μM FUdR was determined in JE6.1cells sensitive (solid circle), JE6.1con (open circle) and resistant,JE6.1AZTR (open triangle) to the antiviral effects of AZT as describedin Materials and Methods.

[0127]FIG. 10 is a graph showing that the AZT-FUdR combination inhibitsHIV-1 infection of PBMC. PBMC were infected with HIV-1 in the absence ofdrug (cross) with AZT alone (x), with various concentrations of FUdRalone, [0.005 μM FUdR (open circle), 0.01 μM FUdR (open square), 0.025μM FUdR (open triangle)], or with combinations of FUdR and AZT[AZT+0.005 μM FUdR (closed circle), AZT+0.01 μM FUdR (solid square)AZT+0.025μM FUdR (solid triangle). Panel A, [AZT]=0.001 μM; Panel B,[AZT]=0.01 μM.

[0128] Table 4. Jurkat JE6. 1 cells, Jurkat JE6. 1 cells refractory tothe antiviral effects of AZT (JE6. AZTR) and control JE6.1 cells(JE6.1con) obtained by infection with MLV-neo in the absence of AZT wereinfected with HIV-1 IIIB in the presence of 0.001 μM AZT, 0.01 μM AZT,0.1 μM AZT, 1 μM AZT or 10 μM AZT in the presence of 0.05 μM FUdR, 0.01μM FUdR or 0.025 μM FUdR. IC₅₀ represents the concentration of AZTrequired for 50% inhibition of reverse transcriptase activity at day 6of infection. TABLE 4 AZT/FUdR Susceptibility In Cells Sensitive AndRefractory To The Antiretroviral Activity Of AZT JE6.1 JE6.1_(AZTR)JE6.1_(Con) IC₅₀ Sensitivity IC₅₀ Sensitivity IC₅₀ Sensitivity Treatment(μM) (fold) (μM) (fold) (μM) (fold) AZT 0.3 0.6 0.2 AZT + .005F 0.3 00.003 20 0.2 0 AZT + .01F 0.1 3 0.001 600 0.03 7 AZT + .025F 0.03 10<.001 >600 0.02 10

Early HIV Breakthrough Infection in the Presence of Stavudine

[0129] Recent clinical analyses have emphasized the potential ofprolonged suppression of HIV viremia when antiviral drug combinationsare used (39,42). However, eradication of virus has not yet beendemonstrated and virus regrowth with cessation of antiviral drugs islikely. In addition, the propensity of HIV with resistance to tripledrug combinations (e.g., AZT, 3TC and a protease inhibitor) to emerge isstill unclear. In the face of this uncertainty more detailed informationconcerning the mechanisms contributing to HIV breakthrough in thepresence of antiviral drugs is needed.

[0130] In this report we utilize an in vitro model of HIV infection toprovide two lines of evidence that early HIV breakthrough infection inthe presence of d4T is not a consequence of infection by HIV withgenetic drug resistance. Initial studies demonstrated that the frequencyof HIV infection in the presence of d4T was very similar with severalstocks of virus predicted to have significant differences in geneticheterogeneity. These studies suggested that any pre-existing unselectedd4T resistant HIV in the population of HIV used to rescue thereplication-defective HIV was not detected above the very high level ofinfection occurring with the other virus populations. The fact thatnearly 50% of the cells infected with HIV in the presence of d4T arereadily re-infected in the presence of high concentrations of d4Tprovides additional evidence supporting a high level of infection in theabsence of genetic d4T resistance. These results are not limited torecombinant HIV and were also demonstrated with MLV based viruses aswell as replication-competent HIV.

[0131] A subset of the cells infected with HIV in the presence of d4T donot have a persistent phenotype of being refractory to d4T. They mayhave been infected as a consequence of cell cycle related phenomena,intravirion reverse transcription (40) or other features that are notcharacterized by persistent cellular phenotypic change. The mechanismsunderlying the refractoriness of both of the populations of cells (thosewith and those without persistent refractoriness to the antiviraleffects of d4T) are currently being assessed with metabolic analyses.

[0132] Previous studies have demonstrated that the combination of FUdRwith AZT or d4T has significant antiretroviral activity (41).Furthermore, the combination of FUDR and AZT has potent antiretroviralactivity in cells refractory to the antiretroviral activity of AZT alone(31). These studies demonstrate the capacity to improve the antiviralefficacy of d4T by the addition of drugs such as FUdR with the capacityof interacting with the biochemical mechanisms responsible for AZTand/or d4T metabolic activation.

[0133] Several investigators have noted clinical features which areconsistent with the results presented above. For example, the definitionof genetic changes associated with clinical d4T resistance has been moredifficult than the definition of genetic changes associated with AZTresistance. In addition, the selection of d4T resistant HIV in tissueculture has also more difficult than the selection of AZT resistantvirus. The high level of early infection with drug sensitive virus mightcontribute to these features.

[0134] In summary, we have demonstrated that early HIV breakthroughinfection in the presence of d4T is not a consequence of infection withvirus that is resistant to d4T. As with AZT, infection withdrug-sensitive virus predominates early after the initiation of thedrug. Further analyses of the mechanisms responsible for HIVbreakthrough in the presence of antiviral drugs are essential to effortsto define drug combinations that provide durable suppression of HIVinfection and viremia (42). Clinical studies with FUdr may be warrantedfor selected patients intolerant of or not optimally responsive tocurrent combination antiretroviral regimens containing either d4T orAZT.

[0135]FIG. 11 is a graph illustrating the infection of JE6.1 cell clonespersistently resistant to the antiviral effects of d4T (D4T bulk, D4TR1,D4TR3) and a control clone of JE6.1 cells with HIV-IIIB in the presenceof various concentrations of d4T. RT activity was assayed and comparedwith those for a control infection in the absence of d4T.

[0136]FIG. 12 is a graph showing that D4T-FUdr combination inhibitsHIV-1 infection of PBMCs. PBMCs were infected with HIV-1 in the absenceof drug [cross], with d4T alone (0.01 uM) [X], with variousconcentrations of FUdr alone (0.005 uM FUdr [open circle], 0.01 uM FUdr[open square], 0.025 uM FUdr [open triangle]) or with combinations ofFUdr and d4T (d4T+0.005 uM FUdr [solid circle], d4T+0.01 uM FUdr [solidsquare], d4T+0.025 uM FUdr [solid triangle]).

[0137] Table 5. HIV-gpt produced in COS cells by transfection withplasmids (plasmid derived) is compared with HIV-gpt produced by rescuefrom H9/HIV-gpt cell line after infection with HIV-IIIB and cellsinfected with MLV-neo. TABLE 5 Frequency of HIV-gpt and MLV-neo ColonyFormation in the Presence and Absence of D4T No. of colonies withoutwith Sources of Virus D4T D4T Frequency plasmid derived 4.7 × 10⁴ 80 1.7× 10³ Rescue with HIV 3.4 × 10⁴ 53 1.6 × 10³ MLV-neo 9.2 × 10⁴ 101 1.1 ×10³

[0138] Table 6. Colony formation and persistent resistance after Hela-T4cell infection with MLV-neo virus in the presence and absence of variousantiviral agents. TABLE 6 Colony Formation and Persistent ResistanceAfter Hela-T4 Infection With MEV-neo Virus in the Presence and Absenceof Various Antiretroviral Agents* No. of No. of persistently Drugcolonies resistant colonies Frequency AZT 12  2/12 2.2 × 10⁻⁴ DDI 19 4/19 4.4 × 10⁻⁴ DDC 17  2/17 2.2 × 10⁻⁴ D4T 63 23/63 2.5 × 10⁻³ Control9050  0/15 0

[0139] Table 7. Infection of Jurkat JE6.1 cell clones sensitive andresistant to D4T. TABLE 7 Infection of Jurkat cell clones Sensitive andResistant to D4T* Clone** No Drug 50 uM D4T 100 uM D4T Bulk ++++ ++ + R1++++ ++++ ++++ R3 ++++ +++ ++ R4 ++++ − − R7 ++++ − − R12 ++++ ++++ ++++R14 ++++ + − R15 ++++ ++++ ++++ R16 ++++ + − R19 ++++ − − R20 ++++ +++++++ R21 ++++ ++++ ++++ R22 ++++ ++ + R26 ++++ + − R29 ++++ − − R33 +++++++ + R36 ++++ ++++ ++ C2 ++++ − − C23 ++++ − − C26 ++++ − − C27 ++++ −− JE6.1 ++++ − −

[0140] Table 8. Jurkat JE6.1 cells refractory to the antiviral activityof d4T (D4T bulk, D4TR1, D4TR3) and control JE6.1 cells were infectedwith HIV-IIIB in the presence of 0.001 uM d4T, 0.01 uM d4T, 0.1 uM d4T,1 uM d4T, 10 uM d4T, 100 uM d4T and 1000 uM d4T in the presence of 0.005uM FUdr, 0.01 uM FUdr or 0.25 uM FUdr. The IC50 represents theconcentration of D4T required for 50% inhibition of RT activity on day 6of infection. TABLE 8 AZT/FUdr Susceptibility in Cells Sensitive andRefractory to the Antiretroviral Activity of D4T JE6.1 D4TBuIk D4TR1D4TR3 IC₅₀ Sensitivity IC₅₀ Sensitivity IC₅₀ Sensitivity IC₅₀Sensitivity Treatment (uM) (fold) (uM) (fold) (uM) (fold) (uM) (fold)D4T 0.02 — 3 — 74 — 2 — D4T + 0.005F 0.003  3 0.8 4 0.9 82 0.1 20 D4T +0.01F 0.001 20 0.04 75 0.2 370 0.03 67 D4T + 0.025F 0.0008 25 0.009 3330.01 7400 0.007 286

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[0183] It will be understood that the embodiments described herein aremerely exemplary and that a person skilled in the art may make manyvariations and modifications without departing from the spirit and scopeof the invention. All such modifications and variations are intended tobe included within the scope of the invention as defined in the appendedclaims.

We claim:
 1. A method for treating a human with human immunodeficiencyvirus infection which comprises administering to the human atherapeutically effective amount of a thymidine analog, which analogacts as an inhibitor of viral reverse transcriptase necessary for viralreplication of human immunodeficiency virus, and a thymidylate synthaseinhibitor, or pharmaceutically acceptable salts thereof.
 2. The methodaccording to claim 1, wherein the thymidine analog is selected from thegroup consisting of 3′-azido-3′-deoxythymidine, and D4T.
 3. The methodaccording to claim 2, wherein the thymidine analog is3′-azido-3′-deoxythymidine.
 4. The method according to claim 1, whereinthe thymidylate synthase inhibitor is selected from the group consistingof 5-fluorouracil, 5-fluoro-2-pyrimidone, and floxuridine.
 5. The methodaccording to claim 4, wherein the thymidylate synthase inhibitor isfloxuridine.
 6. The method according to claim 1, further comprising atherapeutically effective amount of a folate antagonist, or apharmaceutically acceptable salt thereof.
 7. The method according toclaim 1, wherein the folate antagonist is selected from the groupconsisting of methotrexate and trimetraexate.
 8. The method according toclaim 1, wherein the folate antagonist is methotrexate.
 9. The methodaccording to claim 1, further comprising a therapeutically effectiveamount of hydroxyurea, or a pharmaceutically acceptable salt thereof.10. The method according to claim 1, wherein the thymidine analog isadministered in an amount from about 5 mg to 250 mg per kilogram bodyweight per day.
 11. The method according to claim 10, wherein thethymidine analog is administered in an amount from about 7.5 mg to 100mg per kilogram body weight per day.
 12. The method according to claim1, wherein the thymidylate synthase inhibitor is administered in anamount from about 0.01 mg to 25 mg per kilogram body weight per day. 13.The method according to claim 12, wherein the thymidylate synthaseinhibitor is administered in an amount from about 0.01 mg to 10 mg perkilogram body weight per day.
 14. The method according to claim 1,wherein the folate antagonist is administered in an amount from about0.05 mg to 25 mg per kilogram body weight per day.
 15. The methodaccording to claim 14, wherein the folate antagonist is administered inan amount from about 0.05 mg to 10 mg per kilogram body weight per day.16. The method according to claim 1, wherein hydroxyurea is administeredin an amount from about 5 mg to 250 mg per kilogram body weight per day.17. The method according to claim 16, wherein hydroxyurea isadministered in an amount from about 7.5 mg to 100 mgper kilogram bodyweight per day.